In general, bioassays or immunoassays are based on the reaction of a bioactive substance (commonly referred to as the "antigen" A.sub.G) with a specific complex conjugate thereof (commonly referred to as the "antibody" A.sub.B), whereby a complex molecule A.sub.G A.sub.B is formed and the amount of A.sub.G (or, vice-versa, A.sub.B) is measured by various techniques. These techniques belong to two broadly classified groups of methods: the methods involving using an excess of reagent (A.sub.B) and measuring said excess after reaction completion and the methods called the "limited reagent" or "saturation assay" methods (see, for instance, "Radioimmunoassay and Related Procedures in Medicine", Proceedings of a Symposium, Berlin 1977, Vol 1, p. 247 and followings). Briefly summarized, "saturation assays" involve the use of a system in which the test substance or analyte (containing the antigen to be measured) is treated with a limited amount of a specific reagent to give the analyte/reagent complex plus some residual analyte. Yet, prior to the reaction, a known amount of labelled antigen is added to the sample to be tested so that the proportion of the labelled antigen (A.sub.G *) to the unlabelled one (unknown) remains the same in said residual analyte as it was at the start. Since the known amount of A.sub.B used will bind a known amount of the A.sub.G+A.sub.G * mixture it suffices to determine the residual A.sub.G * (by means of its label) to calculate the amount of A.sub.G originally present in the sample. To give an example, suppose that the sample contains x equivalents of an enzyme (A.sub.G) to be measured by means of a known amount (g) of a specific enzyme antibody to this (A.sub.B) that forms an A.sub.G A.sub.B complex (with, for instance, a 1:1 molecular ratio of both components). Then, prior to the reaction, a equivalents of the same enzyme to be measured but in labelled form (A.sub.G *) are added to the sample. Thus, in the course of the reaction, a portion of g equivalents of antigen (A.sub.G +A.sub.G *) is consumed by the g equivalents of antibody. Now, after removing the complex from the mixture, the residual A.sub.G * is ascertained by conventional means. If it is found, by substracting the value measured for the remaining A.sub.G *, that the amount actually consumed was b equivalents, it becomes evident, since A.sub.G and A.sub.G * are chemically indentical and consumed at the same rate, that the ratio of consumed A.sub.G * to consumed A.sub.G, i.e. b/g-b should be equal to the original ratio a/x, from which x=[a(g-b)]/b can be easily calculated. Obviously, the antibody (A.sub.B) and antigen (A.sub.G) can be replaced by any other bonding pairs such as avidin-biotin or vitamin B12-intrinsic factor and members of these conjugate pairs may be determined in an analogous fashion to these described herein. (Exhaustive description of the general features of bioassays in the sense of the present invention can be found in the following references U.S. Pat. No. 4,256,834 and 4,238,565.)
The aforesaid method is quite attractive but however suffers from several drawbacks: First, it requires that the substance to be ascertained be available in pure labelled form, for instance in radioactive form or with other labelling groups (color generating groups, fluorescent groups, specifically chemically reactive groups or light scattering particles). Such labelled compounds may be chemically unstable or short lived (for instance I.sup.131 labelled compounds have a half-life of only 3 months). Second, the test requires that the complex A.sub.G A.sub.B be isolated in pure form (whether the activity of the labelled portion be measured on the complex itself or in the residual mixture) and such separation may be tedious and expensive. Third, labelled A.sub.G * may have, depending on the type of labelling (e.g. by additional groups), a reactivity that may slightly differ from the non-labelled A.sub.G (at least regarding that reaction involving the formation of the analytical complex sought) which is a source of errors in the measurements.
Hence, ideally, a test for achieving the above purpose should simultaneously be specific, sensitive, give inherently accurate results, work under homogeneous conditions and involve storage stable reagents. Consequently, labelling is fundamentally undesirable and a test involving no such labelled reagents should desirably be made available. Such kinds of test already exist based on the experimental fact that, in some case, the A.sub.B and A.sub.G having each more than one reactive site, they tend to form aggregates that will eventually provide light scattering or absorbing effects. Such effects can be measured by conventional nephelometric or colorimetric techniques but, in general, the tests lack sensitivity. The present invention remedies such drawbacks. It comprises contacting with the sample to be determined a substrate the surface of which is, at least partly, coated with a film of or containing A.sub.B (or A.sub.G) distributed on the surface of said substrate, measuring the rate of optical change involved as the result of the reaction of A.sub.B (or A.sub.G) and the A.sub.G (or A.sub.B) of the sample, then correlating the rate curve thus obtained with standard rate curves from identical measurements done with standard samples of A.sub.G (or A.sub.B). Preferably, the optical changes to be measured are those occurring when reflection takes place at the boundary between substrate and film.
By correlating the standard curves with the experimental curves, the desired results about the amount of A.sub.G (or A.sub.B) in the analytical sample can be easily determined according to usual means. For instance, the curves can be compared visually and the results are extrapolated therefrom or the calculation can be done by a computer, standard data being stored in the memory thereof. Thus, if the rate curves are recorded by some automatic measuring and recording equipment, the rate data can be automatically fed to such a computer connected to the equipment and the results can be immediately displayed on a display instrument (meter, digital display or chart recorder). Appropriate analysis of the rate data obtained allows the discrimination of the required reaction between the A.sub.G and A.sub.B from other reactions proceeding at a faster or slower rate and provides a measurement in a shorter time than the same reaction proceeding to equilibrium. In other words, rate curves may be the result of two or more simultaneous processes, the individual rates of which may be distinguished from the slopes of various suitable segments constituting the overall curves. General treatment of complex rate curves is known per se (see for instance, C. P. PRICE & K. SPENCER, Centrifugal Analyzers in Clinical Chemistry, Praeger Scientific (1980), p. 159-169).
In the present invention, the optical changes occuring during the immunoassay reaction involved are preferably monitored by ellipsometry. Although other monitoring techniques may be visualized (see for instance U.S. Pat. No. 4,050,895), ellipsometry has been found, as far, a very sensitive and reliable means for determining the optical changes due to the reaction of the antibody coated surface with the antigen containing sample (see for instance French Pat. No. 2.301.824). Although the exact nature of the complex forming reaction of A.sub.B plus A.sub.G on the film has not been particularly investigated in all cases where the present method is applicable (and actually does not need to be so to provide useful results), it is thought that the said changes in the film are related to thickness and/or refraction index modifications. This is very likely since, indeed, the reaction of the coated A.sub.B and the A.sub.G of the sample logically leads to the conversion of the A.sub.B coating into an A.sub.B A.sub.G complex coating. Thus, ellipsometry is particularly suited in the case.
In order for the reader to better understand how the present invention is implemented practically, some elementary principles of ellipsometry will be briefly reviewed hereinbelow with the help of the accompanying drawing.